Evaluation of the Effect of Two Different Systemic Doses of Viola Odorata on Prevention of Induced Tongue Dysplasia in Rats.

Statement of the Problem Oral cancer is among the ten most common cancers worldwide. It affects the life quality of patients in many ways. Purpose The aim of this study was to compare the effects of two different systemic doses of Viola Odorata syrup on the prevention of 4-Nitroquinoline-1-oxide (4-NQO) induced tongue dysplasia in rats. Materials and Method Forty-eight male Wistar rats were divided into four groups of A, B, C and D. Group A served as the control group. The rats in groups B to D received 30 ppm of 4-NQO in drinking water for 12 weeks. Additionally, the rats in groups B and C received Viola Odorata syrup at doses of 15 and 5 ml/kg, respectively, 3 times a week. Body weights were measured three times a week. At the end, the rats were euthanized and the tongue was removed. Histological evaluations for carcinogenesis were carried out under a light microscope. Results The mean body weight of the rats in groups B, C, and D were lower than that in group A (p< 0.01). After 12 weeks of treatment, microscopically no histological changes of the tongue base epithelia were observed in the control group. The rats in group B did not show severe dysplastic changes; only mild to moderate histological changes including hyperplasia and hyperkeratosis were evident. These incidences were significantly more apparent in groups C with moderate to severe changes (p< 0.05) and group D with severe dysplastic changes (p< 0.01). Almost all rats in group D had hyperplasia and manifested all of the stages of dysplasia. Conclusion Viola Odorata extract has dose-dependent inhibitory effects on the development of tongue induced dysplasia.


Introduction
Oral cancer is among the ten most common cancers worldwide with a multi-factorial cause. [1] The 5-year survival rate is directly related to the stage of disease at diagnosis. Therefore early preventive measures and treatment modalities can decrease the incidence and progression of the disease. [2] Moreover, since many complications of oral cancer treatments such as xerostomia and mucositis affect the life quality of patients, finding an adjuvant medication for the improvement of patient condition seems necessary. [3] Free oxygen radicals such as super oxide, hydrox-yl, and hydrogen peroxide are influential in the incidence of malignancies like oral cancer. These oxidative agents cause DNA breakdown and augment the expression of proto-oncogenes and the damage of tumor suppressor genes. [4] One of the predominant elements of oxidative reactions is inflammation. In presence of inflammatory factors, the production of oxidative agents such as nitric oxide and free oxygen radicals are increased. [3] Antioxidants are agents that destroy the consequence of free radicals in two ways; they can prevent the production of free radicals in the human body, and if produced they can decrease their effects in the body.
Antioxidants can improve the function of the immune system and increase the defensive abilities of the body and therefore, decrease the incidence of cancer and infections. [3] The employed antioxidants are either natural (extracted from animals and plants) or synthetic. In recent years, concerning health issues, great deal of attention has been given to natural antioxidants. Extensive research has been carried out to apply these agents in food products instead of synthetic antioxidants. [5] It is important to note that natural antioxidants do not have the disadvantages of synthetic antioxidants, for example cases of severe bleeding in organs like pancreases has been reported in laboratory animals for synthetic antioxidants. Besides, they can also help maintain the health of individuals when used. [6] Moreover, it is confirmed that the biological nature of traditional and herbal medicine is more compatible with the human body and has fewer side effects. Therefore, currently in many countries around the world, traditional medicine, especially herbal medicine is used for prevention and treatment of diseases. [7] Iran has a climate rich in plant coverage and owing to this fact, antioxidants with a natural source have been given much attention. One of these agents that are being produced in Iran is Odorata syrup extracted from the plant Viola Odorata. [8] The mechanism of action of this agent is to damage and break down the cell wall of cancerous and anti-inflammatory cells. Many laboratory (animal and cellular) and clinical studies have been conducted on this agent that confirm the anticancerous effects of Odorata. Improvement of life quality in patients with malignancies and positive outcomes with no side effects are other benefits verified for this medicinal plant. [8][9][10] Recent studies revealed the dominant effects of cyclotides of Viola Odorata as anticancer agents on gastric, intestinal, rectal, breast, uterine, joints, and pharyngeal cancer. It is reported that its conjunction with chemotherapy can increase the survival rate of patients. [9] Additionally, in cases that chemotherapy is contraindicated, this agent can be used as an alternative with no special care. [9][10] The indication of this agent according to the stud-   Iran).

Methods
The Wistar rats were randomly allocated into four

Results
In the present study 7 of the experimented rats died during the evaluation; 2 from group B, 2 from group C, and    p<0.001, b p<0.01 and c p<0.05).
dysplasia. The incidences of such lesions are listed in Tables 1 and 2  oncology is to add adjunctive treatment modalities to aggressive customary treatments and therefore reduce the destructive effects of usual cancer therapies. [15] Many organic substances have been examined for their antitumor effects in oral cancer. [16][17][18][19] The sweet violet or Viola Odorata is available in permeabilization. Interestingly, CyO2 does not produce significant membrane disruption in primary human brain endothelial cells; this suggests that cyclotides are specified to induce pore formation only in highly proliferating tumor cells. [9,11] Our study showed that Viola Odorata extract has potential anticancer effects in tongue dysplasia of rats.
The anticancer effects of this plant derivative have been investigated, [9,21] but no previous reports exist on the effects of Viola Odorata on oral cancer.
In the present study, the effect of Viola Odorata on the prevention of tongue dysplasia in rat was evalu- In the current study, a difference was seen from mild dysplasia to moderate dysplasia in groups receiving 15mg/kg and 5mg/kg of medication, respectively.
This confirms the importance of dosing and hoping for preferable effects with higher doses of medication. But, it should be pointed out that all substances have side effects which are more prevalent when used in higher doses. It is clear that more animal studies which compare different doses are necessary in order to determine the optimal dose to prevent oral cancer and evaluate possible side effects, paving the way for human studies.

Conclusion
Based on the results of the present animal study, Viola-Odorata has dose-dependent inhibitory effects on the development of dysplasia in tongue. The higher the dose administered the greater the effect of dysplastic prevention. Nonetheless, future animal and human studies seems to be necessary to find the most effective dose.